Method for using ergoline compounds to effect physiological and pathological functions at the 5-HT7 receptor

ABSTRACT

The present invention provides a method for altering mammalian central and peripheral physiological and pathological functioning which is mediated by serotonin binding to the 5-HT 7  receptor. Specifically, the invention provides a method for antagonizing serotonin binding to the 5-HT 7  receptor.

This application is a 371 of PCT/US96/03807 filed Mar. 20, 1996,published as WO96/32944 Oct. 24, 1996.

BACKGROUND

Serotonin mediated regulatory effects are pervasive throughout mammalianperipheral and central physiological functioning. These effects aremediated through binding of serotonin to multiple serotonin receptors atvarious locations in the mammalian body. Serotonin receptors in themammalian body comprise different serotonin receptor subtypes. Serotoninbinding to different receptor subtypes can cause different and oftentimes inimical effects. Hence, in addition to the amount of serotoninreleased and the physiological/pathological status of a receptor, theclinical result of serotonin mediation is also affected by serotoninbinding to more than one receptor subtype.

Presently, there are known to be at least 14 mammalian serotoninreceptor subtypes. Recently researchers identified a novel serotoninreceptor subtype designated as the "5-HT₇ " receptor. mRNA for thisreceptor has been shown to exist in the mammalian central nervous system(CNS), kidney, vasculature, and various regions of the gastrointestinaltract. A homologous 5-HT₇ receptor has also been identified in thecanine coronary artery. The presence of this unique receptor in the CNSand various peripheral smooth muscle tissues, provides the possibilityfor new therapeutic and diagnostic modalities through agonism andantagonism of the 5-HT₇ receptor.

Some compounds which bind to the 5-HT₇ receptor are well known in theart. In fact, binding studies of various known agonist and antagonistcompounds were used to characterize the 5-HT₇ receptor. See Yong Shen etal., "Molecular Cloning and Expression of a 5-Hydroxytryptamine₇Serotonin Receptor Subtype" The J. of Biol. Chem., 264(24):18200-18204(Aug. 24, 1993) hereinafter "Shen"!; Timothy W. Lovenberg et al., "ANovel Adenylyl Cyclase-Activating Serotonin Receptor (5-HT₇) Implicatedin the Regulation of Mammalian Circadian Rhythms", Neuron, 11:449-48(Sept. 1993) hereinafter "Lovenberg"!; Jonathan A. Bard et al., "Cloningof a Novel Human Serotonin Receptor (5-HT₇) Positively Linked toAdenylate Cyclase", The J. of Biol. Chem., 268(31):23422-426 (Nov. 5,1993) hereinafter "Bard"!. Furthermore, the homology of the mammalian5-HT₇ receptor was determined by comparison of the binding affinity ofvarious known serotonin agonist and antagonist compounds to the 5-HT₇receptor in the human, rat, and canine.

In addition to compounds which are known to bind to the 5-HT₇ receptor,the use of ergoline compounds to block non-5-HT₇ receptors is known inthe art. However, ergoline compounds which provide selective highaffinity binding to the 5-HT₇ receptor to cause the central andperipheral physiological effects disclosed herein have not beendescribed previously.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1. (Upper panel) Concentration-relaxation response to 5-HT in theabsence and presence of compound II (0.3-10 μM). (Lower panel) Schildplot for the data from the upper panel. The slope of the Schildregression was 0.92 and the pA₂ was 6.5.

FIG. 2. Concentration-relaxation response to 5-HT in the absence andpresence of compound V (1 μM).

FIG. 3. Concentration-relaxation response to 5-HT in the absence andpresence of compound III (1 μM).

FIG. 4. (Upper panel) Concentration-relaxation response to 5-HT in theabsence and presence of compound IV (10-100 nM). (Lower panel) Schildplot for the data from the upper panel.

FIG. 5. (Upper panel) Concentration-response curve for 5-HT-inducedcontraction in endothelium denuded canine coronary artery under basaltone in the absence and presence of compound IV (1 μM). (Lower panel)Concentration-response curve for 5-MeOT-induced contraction inendothelium denuded canine coronary artery under basal tone in theabsence and presence of compound IV (1 μM)

FIG. 6. (Upper panel) Concentration-response curve for 5-CT-inducedcontraction in endothelium denuded canine coronary artery under basaltone in the absence and presence of compound IV (1 μM). (Lower panel)Concentration-response curves for 5-CT-, 5-HT- and 5-MeOT-inducedcontraction endothelium denuded canine coronary artery under basal tonein the presence of compound IV (1 μM). Data are taken from FIG. 5 (upperand lower panels) and this figure's upper panel.

FIG. 7. Concentration-response curve for sumatriptan-induced contractionin endothelium denuded canine coronary artery under basal tone in theabsence and presence of compound IV (1 μM).

SUMMARY OF THE INVENTION

The present invention provides a method for altering mammalian centraland peripheral physiological and pathological functioning which ismediated by serotonin binding to the 5-HT₇ receptor. Specifically, theinvention provides a method for antagonizing serotonin binding to the5-HT₇ receptor.

In one embodiment, the invention provides a method for treating a mammalaffected with a peripheral condition which is responsive to antagonismof a 5-HT₇ receptor. This includes conditions ameliorated by blockingsmooth muscle relaxation caused by serotonin binding at peripheral 5-HT₇receptors. The method comprises administering an effective amount of a5-HT₇ antagonist compound having the general formula of: ##STR1##wherein: R¹ =NH or O;

R² =H or OR³, wherein R³ is a C₁ -C₆ alkyl;

n=0 or 1; and

the pharmaceutically acceptable addition salts thereof.

Peripheral conditions ameliorated by inhibition of serotonin mediatedsmooth muscle relaxation include hypoperfusion conditions such ascardiovascular shock, septic shock, hypotension, hypovolemia and renalhypoperfusion (e.g., renal vascular spasm) and gastrointestinalconditions such as diarrhea, spastic colon, irritable bowel syndrome(IBS) and inflammatory bowel disease (IBD).

In another embodiment, the invention provides a method for treating amammal affected with a central condition which is ameliorated byblocking serotonin binding at central 5-HT₇ receptors. The methodcomprises administering an effective amount of a 5-HT₇ antagonistcompound having the general formula of compound I, or a pharmaceuticallyacceptable addition salt thereof. Central conditions amenable totreatment according to the method of the invention include circadianrhythm dysfunction, depression, schizophrenia, dementia, migraineheadaches and sleep disorders.

Finally, the method of the invention provides a new and useful tool forfurther probing the role of serotonin in mammalian physiological andpathological functioning.

DETAILED DESCRIPTION OF THE INVENTION

The invention utilizes ergoline compounds as high affinity competitiveantagonists which are selective for the 5-HT₇ receptor. The ergolinecompounds useful according to the present invention are of the generalformula: ##STR2## wherein: R¹ -NH or O;

R² =H or OR³, wherein R³ is a C₁ -C₆ alkyl;

n=0 or 1; and

the pharmaceutically acceptable acid addition salts thereof.

As used herein the term "high affinity" means a binding affinity whichis greater than 100 nM. According to the invention, binding affinity maybe measured in terms of K_(D), K_(i), K_(B), -log K_(B), pA₂ or othervalue used by those skilled in the art.

As used herein, the term "selective" means a compound having relativelyslight affinity for adrenergic receptors such as α₁, α₂, β, histamine,carbachol and like receptors. The high affinity selective binding to the5-HT₇ receptor, by the compounds employed by the invention, provide highpotency antagonism of serotonin at the 5-HT₇ receptor as measured byblockade of relaxation of coronary artery responses to serotoninmediated by 5-HT₇ receptors.

As used herein, the term "condition" means any pathological ornon-pathological syndrome, sign, symptom or physiological event fromwhich a change is desired or beneficial to a mammal. The term"peripheral" refers to physiological or pathological conditions whichare localized outside the central nervous system. The term "central"refers to physiological or pathological conditions which are localizedto the central nervous system (CNS). And, while there is some debate bythose of skill in the art as to the vascular versus neuronal origin ofmigraines and other headaches, for the purposes of the presentdisclosure such conditions are considered conditions of central origin.

In the present disclosure, guidance with respect to conditions amenableto treatment according to the method of the invention is provided ineach instance by representative conditions. It is not meant, however,that the lists recited are exclusive.

Ergoline Compounds

Ergoline compounds employed by the present invention may be prepared bya variety of procedures well known to those of skill in the art. Methodsfor preparation of the compounds of the invention are disclosed in U.S.Pat. Nos. 4,714,704; 4,931,447; 4,981,859; 5,043,341 and 5,141,944 whichare incorporated herein by reference. It is known in the relevant artthat the compounds of the invention comprise one or more asymmetricalcarbons. Accordingly, the method of the invention encompasses individualdiastereoisomers and geometrical isomers as well as racemates of thedisclosed compounds.

The invention also encompasses all pharmaceutically acceptable additionsalts of the disclosed compounds. Pharmaceutically acceptable acidaddition salts of the compounds of the invention include salts derivedfrom inorganic acids such as hydrochloric acid, nitric acid, phosphoricacid, sulfuric acid, hydrobromic acid, hydriodic acid, phosphorous acidand similar inorganic acids. According to the invention,pharmaceutically acceptable acid addition salts also includes saltsderived from organic acids such as aliphatic mono- and dicarboxylicacids, phenyl-substituted alkanoic acids, alkanedioic acids, aromaticacids, aliphatic and aromatic sulfonic acids, and similar organic acids.Such pharmaceutically acceptable salts thus include sulfate,pyrosulfate, bisulfate, sulfite, bisulfite, nitrate, phosphate,monohydrogenphosphate, dibydrogenphosphate, metaphosphate,pyrophosphate, chloride, bromide, iodide, fluoride, acetate, propionate,caprylate, acrylate, formate, isobutyrate, caprate, heptanoate,propiolate, oxalate, malonate, succinate, suberate, sebacate, fumarate,maleate, mandelate, butyne-1.4-dioate, bexyne-1,6-dioate, benzoate,chlorobenzoate, methoxybenzoate, phthalate, terephthalate,benzenesulfonate, toluenesulfonate, chlorobenzenesulfonate,xylenesulfonate, phenylacetate, phenylpropionate, phenylbutyrate,citrate, lactate, β-hydroxybutyrate, glycollate, malate, tartrate,hippurate, lactobionate, methanesulfonate, propanesulfonate,naphthalene-1-sulfonate, naphthalene-2 sulfonate and like salts.

Preferred pharmaceutically acceptable acid additions salts are thoseformed with mineral acids such as hydrochloric acid.

The pharmaceutically acceptable acid addition salts of the compounds ofthis invention can also exist as various solvates, such as with water,methanol, ethanol, dimethylformamide, and the like. Mixtures of suchsolvates can also be prepared. The source of such solvate can be fromthe solvent of crystallization, inherent in the solvent of preparationor crystallization, or adventitious to such solvent. The hydrates areparticularly useful, especially those of the hydrochloride salts.

The 5-HT₇ Receptor

Serotorin effects throughout the mammalian body are mediated throughserotonin binding to serotonin receptors. The ergoline compoundsemployed by the present invention are useful in the treatment ofconditions responsive to inhibition of serotonin binding at 5-HT₇receptors located throughout mammalian central and peripheral tissues.There are several known serotonin receptor subtypes. These receptors aregenerally referred to as "5-HT.sub.(x) receptors" wherein "x" is anumber or a number and letter combination which is used to identify aspecific serotonin receptor subtype. A central or peripheralphysiological or pathological effect caused by serotonin receptoractivation is dependent, in part, on which serotonin receptor subtypeserotonin binds.

In the human, the 5-HT₇ receptor is found in multiple tissue locations.Using reverse transcription PCR techniques, high levels of 5-HT₇ mRNAwas found in the brain, coronary artery and various regions of thegastrointestinal tract (including the stomach, descending colon, andileum). Bard at 23425. High levels of 5-HT₇ receptors in the humancoronary artery and gastrointestinal tract is consistent with theputative smooth muscle relaxant role of 5-HT₇ receptors. Bard at 23425.

The pharmacological profile of the human 5-HT₇ receptor as determined bycompetition of high affinity ³ H!5-HT binding is 5CT>5 MeOT≧5HT>8-OHDPAT wherein 5CT=5-carboxamidotryptamine, 5 MeOT=5-methoxytryptamine,5HT=5-hydroxytryptamine (serotonin) and 8-OHDPAT=8-hydroxy-2-(di-n-propyl-amino)-tetralin. Bard at 23423. Apredominate characteristic of the 5-HT₇ receptor which is seen above,and is consistent with all mammalian 5-HT₇ pharmacological profilesstudied, is the greater affinity of 5CT over serotonin for the 5-HT₇receptor.

According to the method of the invention, it has been discovered thatthe compounds disclosed herein provide high affinity selective bindingto the human 5-HT₇ receptor. High affinity binding to the human 5-HT₇receptor was determined using receptor binding studies as described inJ. M. Zgombick et al., "Expression and Pharmacological Characterizationof a Canine 5-hydroxytryptamine_(1D) Receptor Subtype" Mol. Pharmacol.40:1036-1042 (1991). The general chemical structure for threeparticularly useful compounds of the invention (compounds III-V) areshown below: ##STR3##

The K_(D) and --logK_(B) value for compound II, a known 5-HT₇antagonist, and two compounds employed by the present invention,compound III and IV, are shown in Table 1.

                  TABLE 1    ______________________________________    Antagonist      K.sub.D  (nM)                               -log K.sub.D    ______________________________________    COMPOUND II     609 nM     6.2    COMPOUND III    27.1 ± 1.5 nM                               7.6    COMPOUND IV     13.3 ± 1.2 nM                               7.9    ______________________________________

As indicated in Table 1, antagonist compounds of the present invention(compounds III and IV) have a high binding affinity to the human 5-HT₇receptor. The discovery of the high affinity binding of these compoundsto the human 5-HT₇ receptor provides a new method for the treatment ofhuman central and peripheral physiological and pathological conditionsresponsive to antagonism of serotonin at the 5-HT₇ receptor.

A known effect of serotonin activation of peripheral 5-HT₇ receptors issmooth muscle relaxation. Therefore, in one embodiment, the method ofthe invention provides for treating a human with a condition which isresponsive to inhibition of serotonin mediated smooth muscle relaxationat the 5-HT₇ receptor. This includes, but is not limited to, vascularand gastrointestinal conditions. According to the invention,administration of an ergoline compound of the invention is useful fortreating conditions ameliorated by inhibition of 5-HT₇ mediated vascularrelaxation including systemic hypoperfusion conditions such as septicshock, cardiovascular shock, hypotension, hypovolemia and localizedhypoperfusion conditions, for example, renal hypoperfusion (e.g., renalvascular spasm). Conditions ameliorated by inhibition of 5-HT₇ mediatedgastrointestinal smooth muscle effects include, but are not limited to,gastrointestinal conditions such as diarrhea, spastic colon, IBS andIBD.

In another embodiment, the invention provides for treating a humanaffected with a condition responsive to antagonism of serotonin bindingto central 5-HT₇ receptors which includes, but is not limited to,circadian rhythm dysfunction, depression, schizophrenia, dementia,headaches, including migraines, and sleep disorders.

The pharmacological profile and localization of the 5-HT₇ receptor foundin human tissues is analogous to that found in the rat. Thepharmacological profile of the rat 5-HT₇ receptor as determined bycompetitive binding studies of the nonselective 5-HT receptor antagonistlysergic acid diethylamide ( ¹²⁵ I!LSD) (Lovenberg at 451, Shen at18202) and ³ H!5-HT (Shen at 18202) is 5CT>5MeOT≧5HT>8-OH-DPAT.Lovenberg at 451. Significant to the rat 5-HT₇ receptor system, andanalogous to the human 5-HT₇ pharmacological profile, is the higherbinding affinity of 5CT over serotonin. Moreover, like the human 5-HT₇receptor, the rat 5-HT₇ receptor is positively coupled to adenylatecyclase. Bard at 23423, Lovenberg at 451, Shen at 18203.

The role of serotonin in behavioral and psychological functioning iswell known. The effect of serotonin in resetting or causing phase shiftsof circadian rhythms of neuronal activity in the suprachiasmatic nuclei(SCN) is also known in the art. Lovenberg at 449. In humans, disturbanceof circadian rhythmicity causes mental fatigue and depression. Id. at449. Because 5-HT₇ mRNA in the rat is located in the hypothalamus,particularly in neurons surrounding and possibly within the SCN, it ishypothesized that the 5-HT₇ receptor is responsible for the knownserotonergic mediation of circadian rhythms. Lovenberg at 453.Accordingly, the inventors foresee the use of the compounds of theinvention to inhibit serotonin mediated circadian rhythm phase advances.Hence, this provides a method for treating a human affected with, forexample, jet lag and sleep disorders of a circadian nature.

The 5-HT₇ receptors blocked by the compounds employed by the presentinvention also play a role in psychiatric disorders. In addition to the5-HT₇ receptor being located in various limbic and cortical regions ofthe brain, when expressed in mammalian cells, the 5-HT₇ receptor shows ahigh affinity to the antipsychotic drugs clozapine and loxapine, and thetricyclic antidepressant drug, amitriptyline. Shen at 18200. Therefore,the inventors foresee the use of the invention to treat psychiatricdisorders which are responsive to blockade of serotonin at central 5-HT₇receptors. This includes such conditions as depression, dementia andschizophrenia.

The homology of the human 5-HT₇ receptor and various functionallydefined receptors in the blood vessels of nonhuman species is recognizedin the art. Bard at 23422. The uniqueness of the canine coronaryrelaxant receptor among serotonin receptors described prior tocharacterization of the 5-HT₇ receptor has been discussed by variousresearchers including the inventors. Daniel J. Cushing et al."Serotonin-Induced Relaxation in Canine Coronary Artery Smooth Muscle,"J. of Pharmacol Exptl. Ther., 263(1):123-129 (1992).

The Examples of the present invention provide specific detail forgeneration of pharmacological profiles which support the homology of thecanine coronary artery relaxant receptor and the 5-HT₇ receptor. Likeother members of the 5-HT₇ family of receptors the canine coronaryartery relaxant receptor has a greater affinity for 5CT than for theendogenous agonist serotonin, a hallmark of the 5-HT₇ receptor.

The dissociation constants determined by antagonist effects in thecanine coronary artery for the known 5-HT₇ antagonist, compound II, andthree preferred compounds of the invention (compounds III-V) are shownin Table 2.

                  TABLE 2    ______________________________________    Antagonist      pA.sub.2  (-log K.sub.B)                               (slope)    ______________________________________    COMPOUND II     6.5        0.92    COMPOUND III    6.7    COMPOUND IV     8.3        0.98    COMPOUND V      6.4    ______________________________________     (One-way ANOVA; TukeyKramer; p < 0.05).

As discussed above, the 5-HT₇ receptor has been localized to the humancoronary artery analogous to the canine coronary artery relaxantreceptor. The following Examples provide functional studies whichdemonstrate the use of the invention to inhibit serotonin mediatedvasodilation in tissue samples. Accordingly, the inventors foresee theuse of the invention for treatment of conditions in mammals which areameliorated by blockade of serotonin mediated vascular relaxation.Conditions which may be ameliorated according to the method of theinvention include systemic hypoperfusion conditions, for example, septicshock, cardiovascular shock, hypotension, hypovolemia and localizedhypoperfusion conditions such as renal hypoperfusion.

The discovery of the selective high affinity binding to the 5-HT₇receptor by the compounds employed by this invention also provide auseful diagnostic modality for further understanding of the role ofserotonin in mammalian physiological and pathological functioning.Moreover, discovery of compounds having selective high affinity bindingto the 5-HT₇ receptor is fundamental to production of therapeutic agentsof high therapeutic efficacy with minimal side effects.

EXAMPLES Example 1 Tissue Preparation for Isolated Tissue Studies

Hearts were obtained from mongrel dogs anesthetized with sodiumpentobarbital (30 mg/kg, i.v.) and placed in oxygenated modified Krebs'solution of the following composition (millimolar): NaCl, 118; KCl, 4.6;CaCl2, 1.6; KH₂ PO₄, 1.2; MgSO₄ 1.2, glucose, 10; NaHCO₃ 24.8 (pH 7.4).The left anterior descending and circumflex coronary arteries weredissected from the hearts, cleaned of fat and adhering tissue and cutinto rings (approximately 2-3 mm outer diameter and 4-5 mm in length).The intimal surface of the rings was gently rubbed with a pair offorceps to remove the endothelium. Tissues were then mounted between twostainless steel hooks and placed in 10 ml organ chambers constantlyexposed to oxygenated modified Krebs' solution (95% O₂ /5% CO₂ ; pH 7.4;37° C.). Tissues were equilibrated for 1 hour under an initial optimalpassive force of 4 g determined previously from length-tensionexperiments. The buffer was replaced every 15 minutes duringequilibration. Isometric force was measured with Statham UC3 forcetransducers connected to a Beckman dynograph (R611). After 1 hrequilibration, tissues were depolarized twice with 50 mM KCl for 30 minwith a 20 min washing period between. The integrity of the endotheliumwas examined with acetylcholine (1 μM), bradykinin (100 nM), or theionophore A23187 (1 μM). Rings that relaxed to these agents were notused in this study.

Example 2 Experimental Protocol and Data Analyses

Tissues prepared as described in Example 1 were precontracted with PGF₂α(10 μM) which produced a tonic contraction that remained at or above92.8±2.1% (n=14) of the original maximum force throughout theexperiment. Cumulative concentration-response curves to 5-HT weregenerated in tissues incubated with either vehicle or antagonist for 1hr. In some experiments contraction was measured in tissues under basaltone upon the cumulative addition of 5-HT. These tissues were treatedwith prazosin (1 μM) to block any potential a adrenoceptor activationand in the absence or presence of antagonist.

All results are expressed as mean ±S.E. where "n" represents the numberof rings examined. No more than two rings were used from each animal foreach compound studied. The relaxation data is expressed as percentrelaxation of the contraction produced by PGF₂α. The contraction data isexpressed as percent of a maximal KCl (50 mM) contraction. The EC₅₀ wasthe concentration of the agonist producing half-maximal response and wasdetermined by least squares linear regression analysis of the linearportion or the agonist concentration-response curve and expressed as-log C₅₀ (pEC₅₀)

Apparent antagonist dissociation constants (K_(B)) were determined foreach antagonist according to the following equation: ##EQU1## where (B)is the concentration of the antagonist and dose ratio is the EC₅₀ of theagonist in the presence of the antagonist divided by the EC₅₀ of theagonist in vehicle treated tissues. These results were then expressed asthe negative logarithm of the K_(B) (i.e. -log K_(B)). Significantdifferences between vehicle and antagonist treated tissues weredetermined with Student's t-test. The alpha value was set a priori at0.05.

The data was also analyzed in accord with the procedure of Arunlakshanaand Schild (1959). The dose ratio was determined at variousconcentrations of antagonist compound II or compound IV. If blockade iscompetitive under equilibrium conditions a plot of the logarithm (doseratio--1) against the negative logarithm of the molar concentration ofantagonist should yield a straight line whose slope is not differentfrom unity and whose intercept on the abscissa is the pA₂, which isgenerally considered to be equivalent to -log K_(B).

Serotonin, acetylcholine, bradykinin, and A23187 were purchased fromSigma Chemical Co. (St. Louis, Mo.). compounds II-V were synthesized inthe Lilly Research laboratories.

Example 3 Effect of Ergoline Esters on 5HT-Induced Relaxation in CanineCoronary Vessels Contracted with PGF₂α

In rings precontracted with PGF₂α (10 μM), compound II (0.3-10 μM)shifted the concentration-response curve to 5-HT significantly to theright and did not alter the maximum relaxant response (FIG. 1, upperpanel). This antagonism was competitive as the slope of the Schild plotwas not significantly different from unity (FIG. 1, lower panel). Inaddition, the pA₂ value for compound II was 6.5 (Table 2).

In rats, after oral administration, compound V has a higherbioavailability than compound II. Compound V (1 μM), like compound II,blocked 5-HT-induced relaxation in an apparently competitive manner(FIG. 2). pEC₅₀ values between vehicle and compound V-treated tissueswere significantly different (p<0.05; Student's t-test). In addition,the -log of the antagonist dissociation constant for compound V at the5-HT₇ receptor in the dog coronary artery was 6.4 (Table 2).

Example 4 Effect of Ergoline Amides on 5-HT-induced Relaxation in CanineCoronary Vessels Contracted with PGF₂α

In addition to examining the activity of the ergoline-esters at thisreceptor, the inventors examined the activity of two ergoline-amides atthis site; compound III and compound IV. After oral administration inrats, compounds III and IV have a greater bioavailability than the estercompounds II and V. Compound III (1 μM) and compound IV (10-100 nM),blocked 5-HT-induced relaxation in endothelium-denuded canine coronaryartery rings in a competitive manner (FIGS. 3, 4). The -log of theantagonist dissociation constant for compound III and compound IV was6.7 and 8.3, respectively. In FIG. 3, pEC₅₀ values between vehicle andcompound III-treated tissues were significantly different (p<0.05;Student's t-test). The slope of the Schild regression in the lower panelof FIG. 4 was 0.98 and the pA₂ was 8.3.

Example 5 Effect of Compound IV on 5-HT-, 5-CT- and 5-MeOT-inducedContraction in Canine Coronary Vessels Under Basal Tone

As previously reported in D. J. Cushing et al., "Comparison of theSerotonin Receptors that Mediate Smooth Muscle Contraction in Canine andPorcine Coronary Artery," J. Pharmacol Exotl. Ther. 856-862 (1992),along with their ability to dilate canine coronary artery, 5-HT and5-MeOT produced a biphasic contractile response in vessels under basaltone (FIG. 5). Compound IV (1 μM) significantly attenuated the relaxantphase of this response while not affecting the contractile responseproduced by both agonists (FIG. 5).

5-CT did not significantly contract coronary arteries either under basaltone (FIG. 6, upper panel) or when precontracted; however, it was apotent relaxant agonist in canine coronary artery and has highestaffinity for 5-HT₇ receptors. In the presence of compound IV (1 μM), toblock 5-CT-induced relaxation, 5-CT concentration-dependently contractedcanine coronary artery (FIG. 6, upper panel). To further document theselectivity of compound IV for the relaxant 5-HT receptor over thecontractile 5-HT receptor in the canine coronary artery we examined theeffect of compound IV (1 μM) on the contraction produced by sumatriptan,an agent lacking relaxant activity in this preparation. In confirmationof its selectivity, compound IV (1 μM) did not significantly affectsumatriptan-induced contraction (FIG. 7).

According to the method of the invention, a compound of the generalformula of compound I is administered orally or parenterally to a mammalhaving a condition ameliorated by antagonism of serotonin binding at aperipheral or central 5-HT₇ receptor. The compounds of the presentinvention have previously been shown to have systemic effects after oraladministration to rats.

For parenteral administration, a water soluble salt of the drug isdissolved in an isotonic salt solution, or other pharmaceuticallyacceptable carrier, and administered by the intravenous, intramuscularor subcutaneous route. For oral administration, apharmaceutically-acceptable salt of the drug is mixed with standardpharmaceutical excipients such as starch and loaded into capsules ormade into tablets, each containing 0.01 to 100 mg of active drug. Dosagelevels of from about 0.01 to 30 mg/kg are effective in blocking 5-HT₇receptors. Thus the oral dosage would be administered 1-4 times per day,giving a daily dosage range of about 0.01 to about 120 mg/kg per day.

The inventors further recognize that with time there may be discovery ofadditional physiological and pathological conditions mediated byserotonin binding to the 5-HT₇ receptor. Hence, in addition to thespecific central and peripheral conditions which may be treatedaccording to the method of the invention, the inventors foresee futuretherapeutic and diagnostic uses for the compounds of the invention intreatment of other effects mediated through the 5-HT₇ receptor.

All publications and patents in this specification are indicative of thelevel of ordinary skill in the art to which this invention pertains. Allpublications and patents are herein incorporated by reference to thesame extent as if each individual publication or patent application wasspecifically and individually indicated by reference.

It will be apparent to one of ordinary skill in the art that manychanges and modifications can be made in the invention without departingfrom the spirit or scope of the appended claims.

We claim:
 1. A method of inhibiting the 5-HT₇ receptor in a mammal,comprising administering to a mammal in need of such inhibition a 5-HT₇inhibiting dose of a compound of formula ##STR4## wherein: R=NH or O;R²=H or OR³ wherein R³ is a C₁ -C₆ alkyl; n is 0 or 1; or apharmaceutically acceptable salt thereof.
 2. A method of claim 1 wherethe mammal is suffering from cardiovascular shock.
 3. A method of claim1 where the mammal is suffering from septic shock.
 4. A method of claim1 where the mammal is suffering from hypotension.
 5. A method of claim 1where the mammal is suffering from hypovolemia.
 6. A method of claim 1where the mammal is suffering from renal hypoperfusion.
 7. A method ofclaim 1 where the mammal is a human.
 8. A method of inhibiting the 5-HT₇receptor in a mammal, comprising administering to a mammal in need ofsuch inhibition a 5-HT₇ inhibiting dose of a compound of formula##STR5## or a pharmaceutically acceptable salt thereof.
 9. A method ofclaim 8 where the mammal is suffering from cardiovascular shock.
 10. Amethod of claim 8 where the mammal is suffering from septic shock.
 11. Amethod of claim 8 where the mammal is suffering from hypotension.
 12. Amethod of claim 8 where the mammal is suffering from hypovolemia.
 13. Amethod of claim 8 where the mammal is suffering from renalhypoperfusion.
 14. A method of claim 8 where the mammal is a human.